Particle adsorption

ABSTRACT

Lipin particles are removed from aqueous suspension by adsorption on hydrophilic macromolecules substituted with pendant hydrophobic groups. Particularly beneficial results are achieved by use of pendant hydrophobes linked by strongly ionogenic groups to water insoluble carriers.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. patent applicationSer. No. 128,617, filed Mar. 10, 1980, now abandoned.

BACKGROUND OF THE INVENTION

This invention is concerned with the separation of lipin-containingparticles from aqueous milieu. More specifically this invention relatesto the removal of lipoprotein or glycolipid-containing vesicles fromaqueous suspensions. This invention is particularly concerned with thegeneral, nonspecific adsorption of microbes such bacteria, yeast, fungiand viruses from contaminated aqueous suspensions.

Lipins are a group of compounds comprising fats and lipoids which aresoluble in ether. They include fats, fatty oils, essential oils, waxes,sterols, phospholipids, glycolipids, sulfolipids, aminolipids,chromolipids, fatty acids and lipoproteins.

A great variety of biological structures contain lipins. For example,particles such as animal viruses may contain lipids at up to about 50percent by weight. Chylomicons, a major circulating lipid transportmedium in higher animals, are essentially fat globules enveloped by alipoprotein membrane. Animal cells, bacteria, yeast and fungi allcontain varying proportions of lipins in their cell walls andprotoplasm.

Chylomicrons, liposomes, cellular microorganisms and animal cells areexamples of lipin vesicles, a major class of lipin-containing particleswith which this invention is concerned. Lipin vesicles are genericallydefined as substantially water insoluble particles ranging about from250 to 10,000 A in mean diameter which are characterized by alipin-containing membranous envelope enclosing a liquid interior. Thecontained liquid may consist almost entirely of lipid, as in the case ofchylomicrons, or be relatively free of the substance, as in the case ofmicroorganisms. It is not necessary that the contained liquid have anylipid content whatsoever.

The presence of such lipin particles in aqueous suspensions haspresented many problems for a great variety of arts. The foremostdifficulty has been encountered with microbial contamination of aqueousliquids intended for administration to living organisms, particularlyparenteral fluids infused into patients. While parenteral fluids arecarefully manufactured so as to be sterile it is common practice forusers to add medications or other additives to the solutions. Thisprovides a potential avenue for contagion to enter the patient's bloodstream. Thus it has been previous practice to modify the parenteralsolution administration sets which are used to provide a controlledfluid flow from the solution container to the patient's vein by theinclusion of a filter capable of physically entrapping cellularmicroorganisms. Similar filters have been used with solutionadministration sets in continuous ambulatory peritoneal dialysis. Suchfilters, hereinafter referred to as sterile filters, are generallyporous membranes having an average pore diameter of about from 0.2 to0.45 microns, ordinarily about 0.2 microns. These filters are capable ofretaining most cellular microorganisms since the smallest bacterium isbelieved to be about 0.3 microns in diameter.

The pharmaceutical industry has also employed membrane filters of thistype to sterile filter products which cannot be chemically or thermallysterilized because of their lability. Examples of such products includeinsulin and human blood protein fractions such as factor VIII.

Sterile filters approach the absolute in retaining particles greaterthan the stated pore size. However they are readily clogged byrelatively small numbers of particles, particularly those which have apore size close to the average pore size of the filter. Consequently, itis conventional to pass the liquid to be sterilized through a depthfilter before contacting it with the sterile filter. These filters havea high capacity to retain particles throughout rather than by sievingonly at the liquid-filter interface.

Depth filters are fabricated from many materials including cellulose,polypropylene, diatomaceous earth and asbestos. Most of the depthfilters trap particles by physical entrapment at points where two ormore fibers or granules form a pore, alone or in combination with whatusually are assumed to be London-van der Waal's attractive forces. Depthfilters have the advantage of removing particles while retaining a highfilter flux, i.e., a high flow rate of feedstock per unit of filtrationarea, even in the face of a particle load that would rapidly clog asterile filter. However, depth filters are largely ineffective inremoving particles in the 0.5 to 3 micron size range, at least whencompared to sterile filters. Of course, viruses are too small to beretained even by sterile filters. A long felt need has existed for aunitary filter which is capable of removing particles having a widerange of sizes from suspension, including particularly bacteria andviruses, without suffering a significant reduction in flux when exposedto heavy particle loads. At the very least, considerable improvementcould be secured by using a depth filter having such characteristics,thus freeing the sterile filter from its role as the virtually sole lineof defense against passage of the smallest cellular organisms andthereby reducing the probability of clogging or flux reductions.

The sterile filters used in parenteral administration sets rarely haveto deal with high levels of suspended particles, and thus clogging isnot usually encountered. However, the flux of hydrocolloidal solutionssuch as blood protein fractions through sterile filters is very low.This low flux is attributed to the affinity of the hydrocolloids for thefilter surfaces, resulting in increased hydrocolloid binding by thefilter and the formation of hydrocolloid concentration gradientsupstream from the filter surface. Similar problems are encountered inindustrial sterile filtration of the same products. Consequently,membrane filters used in conventional parenteral administration sets forlarge-volume, non-colloidal parenterals such as dextrose or proteinhydrolysates in water have proven inadequate for the filtration ofviscous hydrocolloid solutions such as factor VIII or albumin.

Filters having small pores create another problem in parenteral solutionadministration. In the low pressure environment of an infusion anyresidual air in the administration set tubing will accumulate againstthe wetted filter rather than passing through. This phenomenon is termedair-blocking, and frequently it requires that the set be discarded.Efforts have been made to remedy this problem by inclusion of anon-wetting or hydrophobic filter in the set in addition to the normalhydrophilic member (U.S. Pat. No. 4,004,587). This of coursenecessitates the inclusion of two different types of filters in the set,an added expense.

Finally, all filters which rely solely upon the mechanical exclusion ofparticles depend upon the dimensional stability of the particles. If theparticles can deform so as to pass through the micron sized pores of thefilter then the filter will effectively fail. Mycoplasma, which arebacteria devoid of rigid cell walls, are highly deformable. Otherfamilies of microorganisms also exhibit varying degrees of deformationunder pressure, as do mammalian cells and chylomicrons. The filtrationof suspensions of such particles would be more reliable if mechanicalexclusion could be supplemented.

In summary therefore it is apparent that the previous efforts of the artto remove particles from suspension primarily on the basis of mechanicalexclusion has resulted in considerable difficulties.

Clinical chemistry is another art in which lipin particles have createdproblems. Blood samples taken for diagnostic assay of constituents aregenerally permitted to clot and the resulting serum removed byaspiration or decantation, frequently with the aid of devices such asdisclosed in U.S. Pat. No. 3,865,731. While this process removes mostcellular matter from the test sample it fails to reduce the level ofother insoluble particles, most notably chylomicrons. Such particlesinterfere in subsequent optical assays of serum constituents.

An optical assay is defined as any analytical method in which theconcentration or activity of an analyte is measured by a change in lightas it is passed into the sample, and includes nephelometry andspectrophotometry in the main. Lipemic serum samples often containchylomicrons in such concentrations that the serum appears milky, andeven at lower chylomicron concentrations light scattering particles inthe sample will interfere. While such samples may be diluted to reducethe interference this also necessarily dilutes the analyte, therebyreducing sensitivity, and in any case the comparative effect of thechylomicrons is still significant relative to analyte concentration.Reagents such as detergents may be added to destroy the lipid suspensionbut may interfere in various assays (United Kingdom Pat. No. 1,542,982).Ultracentrifugation will remove the particles but requires costlyequipment and is tedious to perform. A need therefore exists for amethod and device to remove lipin particles from biological fluids to beassayed by optical methods.

A need also exists in many arts to nonspecifically remove animal virusesfrom aqueous compositions. While the virions in many aqueous substancescan be inactivated by pasteurization or chemical sterilization, manylabile products, particularly pharmaceuticals and some blood proteinfractions, are sensitive to such harsh treatments. These techniques arealso not suitable for high volume treatments such as drinking waterpurification because of the high cost. Mechanical entrapment of virionsby filtration ordinarily is not practical because at the required poresizes the filter flux is extremely low.

Various investigators have looked into the use of immunoadsorbents toseparate hepatitis virus. However, this technique is of little usebecause supplies of antihepatitis are limited, there is a risk ofleaching antibody into the adsorbent effluent and, primarily, theantibody is necessarily capable of binding only the hepatitis virion andis not effective in removing other harmful viruses.

Immunoadsorbents have also been used in various affinity chromatographytechniques for cell separation. See Cuatrecasas et al., "Ann. Rev.Biochem." 40:275 (1971). In these techniques inert matrices aresubstituted with ligands. Animal cells expected to contain membranereceptor proteins for the ligands are contacted with the immobilizedhaptens. Those cells having receptors specific for the ligand are boundwhile the remainder are washed free of the substrate, thus enabling oneto obtain specific cell lines. Such methods are, however, of no usewhere the object is to remove a diverse cell population from suspensionbecause the receptor sites are unknown and, in any case, would be sonumerous that preparing immobilized ligands for all of them would beimpractical.

This handicap would appear to be shared by the process of U.K. patentspecification No. 1,531,558 to Kabi. This patent discloses adsorbinghepatitis virus from plasma and some solutions of blood proteinfractions with a water permeable matrix having a coupled hydrophobicligand of more than 7 carbon atoms or a condensed ring system. Theadsorbent is disclosed to have a high and specific affinity forhepatitis virus. A need therefore remains for an adsorbent for animalviruses which is not specific for any one virus.

Tanny et al., "J. Parenteral Drug Association" 33(1):40-51 (1979)speculate that 0.45 and 0.20 micron cellulose triacetate membranesretain Pseudomonas diminuta by a combined adsorptive and sieve effect.Similarly, Tanny et al. advance the same hypothesis to account forlosses in the titer of influenza vaccine passed through mixed celluloseesters, cellulose triacetate and acrylonitrile-vinyl chloride copolymer["J. Parenteral Drug Association" 32(6):258-267 (1978)].

Pertsovskaya et al. "Biol. Nauki" 14(3):1005 (1971) disclose that glass,methylene and amine-substituted glass, and films of polyamide,polysacrylate, cellulose triacetate, and polyethylene all adsorbdifferent groups of bacteria to varying degrees. In some cases, e.g.,with bacilli, no adsorption at all was observed. Gerson et al. inImmobilized Microbial Cells, K.Ven Katsubramanian, Editor, pp 39-43(1978) also report adsorbing various bacteria to surfaces.

Ambergard™ filters, which are ion exchange resins having the structureR-N⁺ (CH₃)₃ X wherein R is a styrenedivinylbenzene copolymer and X isOH--, Cl-- or SO₄ =, have been used to upgrade the bacteriologicalquality of demineralized water for ultimate use in pharmaceuticals (Rhomand Haas literature dated June, 1978). In this connection, see U.K.patent application No. 2,009,623A.

U.S. Pat. Nos. 4,007,113 and 4,007,114 to Ostreicher employ a matrix ofself bonding and electronegative fibers having surfaces coated withmalamine-formaldehyde cationic colloid for filtering contaminatedliquids.

Hjerten et al., "J. of Chromatography" 101:281-288 (1974) discloses thatsatellite tobacco necrosis virus and baker's yeast cells are retained incolumns of non-ionogenic hydrophobic agarose in the presence of elevatedsalt concentrations.

Similarly, Magnusson et al. in "Immunology" 36:439-447 (Mar. 19, 1979)disclose that adsorption of S. typhimurium occurs when placed on acolumn in the presence of 1M (NH₄)₂ SO₄, but that the bacteria elute asthe salt concentration is reduced.

Halperin et al., "Biochemical and Biophysical Research Communications"72(4):1497-1503 (1976) disclose desorbing erythrocytes retained on alkylagarose columns by repeated pipetation in the presence of bovine serumalbumin.

Accordingly it is an object of this invention to adsorb a wide spectrumof cells including animal cells, unicellular organisms, bacteria, yeast,fungi and viruses, from aqueous suspensions, using a single adsorbentcomposition.

It is another object to provide improved hydrophobic adsorbentcompositions.

It is a further object of this invention to remove lipin particles fromlipemic body fluids such as serum or plasma and provide an improveddevice therefor.

It is another object to pasteurize alcoholic beverages without the costand detriment to flavor inherent in prior methods.

It is an additional object of this invention to sterile filterparenteral solutions, particularly solutions containing protein or lowconcentrations of salt, at increased flux and with greater assurance ofsterility than heretofore possible, and to provide an improved devicetherefor.

It is another object to provide an improved surface for the cultivationof mammalian cells in tissue culture or for binding enzyme-containinglipin particles used in enzyme reactors.

These and other objects of this invention will be apparent fromconsideration of this specification as a whole.

SUMMARY OF THE INVENTION

It has now been found that hydrophilic moieties having pendanthydrophobic groups and strong ionogenic groups avidly adhere to a greatvariety of lipin particles, including animal virions, animal cells,bacteria, yeast, fungi and chylomicrons. Accordingly, certain of theobjects of this invention are achieved by contacting an aqueoussuspension of lipin particles with novel compositions having the formula

    [(Y).sub.e B].sub.d Z

wherein Y is a hydrophobic ligand, B is a strong ionogenic group, Z is awater insoluble carrier, e is an integer and d is greater than 2, andthen separating the composition from the fluid.

Further, it has been found that adsorbent compositions having theformula [(Y)_(e) B]_(d) Z, wherein Y, Z, e and d are as described aboveand B is a linking group or bond, are capable of nonspecificallyadsorbing aerosols of lipin particles or such particles from aqueousliquids containing proteins, ethanol or low ionic strength.

In addition, such adsorbent compositions have been found useful inmammalian tissue culture and as a binding medium for the adsorption oflipin vesicles having active enzymes for use in enzyme reactors.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The hydrophobic ligand Y is characterized broadly by its low solubilityin water and its affinity for lipid solvents, primarily ether. Suitablehydrophobes are generally those in which, when the group BZ is replacedby methyl, the water solubility of the resulting compound at 20° C. willbe less than about 0.075 parts by weight of the compound per 100 partsby weight of water and its solubility in ether at the same temperaturewill be infinite. Preferably this hypothetical compound will beinsoluble in water in 20° C. Its molecular weight will range about from70 to 600, ordinarily about from 100 to 400. On a molecular level thependant hydrophobe should roughly appear as a cylinder having averagedimensions of about from 7 to 40 A in length and about from 3 to 15 A;preferably about from 3 to 10 A, in diameter. The effect of thehydrophobe is generally less satisfactory as the diameter increasesabove about from 10 to 15 A, but the length is less material.

The hydrophobe-substituted adsorbents herein generally contain pendanthydrophobic ligands having the formula ##STR1## and wherein R ishydrogen, nitro, alkyl, alkyl ether, halogen, monocyclic aromatichydrocarbon or a carbocycle system;

A is a bond, monocyclic aromatic hydrocarbon or carbocycle system;

b is an integer;

J is oxygen, sulfur or a bond; and

n and y are zero or an integer;

with the proviso that where A is a bond and R is hydrogen, nitro orhalogen then the sum of n and y is an integer greater than 5.

Suitable carbocycle systems for the hydrophobe adsorbents are multiplehydrocarbon ring systems which may be fused or bridged, contain aboutfrom 4 to 30 carbon atoms and be saturated or unsaturated. Preferablythe systems will contain about from 6 to 20 carbon atoms and be eitheraromatic or fully saturated. Examples of suitable bridged systems arebicyclo[2.2.1]heptane, bicyclo[3.2.1]octane, bicyclo[1.1.0]butane andbicyclo[2.2.1]hept-2-ene. Suitable spiro systems are spiro[2.3]pentaneand spiro[3.4]oct-1 ene. The fused systems may be ortho or peri,preferably ortho such as naphthalene, indene, fluorene, arthracene andphenanthrene. Ortho fused systems having more than 3 rings, e.g.steroids such as cholesterol, may also be employed. Ring assemblies suchas tercyclohexane and biphenyl are acceptable.

The monocyclic aromatic hydrocarbons useful in or as the pendanthydrophobic ligand will contain generally about from 6 to 12 carbonatoms, preferably 6-8 and are most desirably phenyl.

All R groups are preferably hydrogen, although substitution with nitro,alkyl, alkyl ether, halogen, monocyclic aromatic hydrocarbon orcarbocycle systems is within the scope of this invention. Ordinarily,about from 1 to 3 R groups will be other than hydrogen, alkyl or alkylether. Suitable halogens are fluorine, chlorine or bromine, preferablyfluorine. The monocyclic aromatic hydrocarbons or the carbocycle systemsare usually singly substituted at the pendant hydrophobe terminus, witha monocyclic aromatic hydrocarbon preferred over a carbocyclic system.The branched chain systems which result from the use of alkyl R groupsare generally satisfactory where R is a short chain alkyl or alkylether, on the order of C₁ to C₆.

Group J is preferably a bond. If J is other than a bond then the oxygenether is preferred.

Generally n+y will range from 1 to about 20 in total, but each will tendto vary inversely with one another if X is oxygen or sulfur. The sum ofn and y is usually about from 4 to 25, preferably 7 to 23 where A is abond and R is H. The value for n+y preferably will be about from 1 to 10it at least one R is an aromatic hydrocarbon or carbocycle system, andparticularly when A is a bond and not a ring. Where J is oxygen orsulfur, n is usually greater than 2, particularly when no terminal R isan aromatic hydrocarbon or carbocycle system. Where A is a bond and R ishydrogen, nitro or halogen then the sum of n and y is greater than 5.

The number of Y groups, designed e, will depend upon the nature of thelinking group chosen. Generally, e will range from 1 to about 3, withone being preferred.

The degree of major branching of the hydrophobic ligand is designed byb. In the preferred instance both b and e are 1 when A is a bond. When Ais an aromatic hydrocarbon or carbocycle system the designation bindicates the degree of substitution of the aromatic hydrocarbon orcarbocycle system. This degree of substitution is preferably low, with branging about from 1 to 4. This is particularly the case where an Rgroup is an aromatic hydrocarbon or carbocycle system, or where n+y isgreater than 5.

The preferred hydrophobes are long chain, normal, secondary or tertiaryalkyl such as n-hexyl, n-octyl, n-dodecyl, n-tetradecyl, or n-octadecyl.

The degree of substitution of the hydrophilic macromolecule with pendanthydrophobic groups is represented by d, a minimum of 2 with a maximumdependent upon the characteristics desired in the adsorbent. The degreeof substitution must be correlated with the size of the macromolecule Z,its hydrophilicity, i.e., the nature of the non-hydrophobic substituentsof Z, the hydrophilicity of the linking groups B and the dimensions ofthe hydrophobe ligand. The adsorbent as a whole should be water wettablebut not water soluble. Accordingly, d should not be so high as to renderthe material water repellant. Generally, the ratio of d to the number ofhydrophilic groups that exist on the hydrophilic macromolecule at thepH, temperature and ionic conditions of adsorbent use, i.e., the numberof exposed polar groups, will range about from 2:1 to 1:50. Morespecifically, when Z is an organic, hydrophilic polymer then d willrange about from 0.5 to 0.1 times the number of monomer unitsconstituting the polymer.

The BZ moiety functions as a hydrophile. The linking groups B, the waterinsoluble carrier Z, or both B and Z contain hydrophilic groups whichimpart several desirable properties to the adsorbent. First, permeationof aqueous suspensions into adsorbent matrices is facilitated by theoverall water wettability of the matrices. Second, the adsorbentaffinity for lipin particles is enhanced by the net hydrophiliccharacter of the adsorbent when compared to entirely hydrophobicsurfaces or those in which the hydrophobe is not pendant, i.e., branchedfrom a hydrophilic matrix. Third, the presence of hydrophilic groupslessens the nonspecific binding of low molecular weight lipophiliccompounds such as drugs and dyes. Fourth, the combined effect of ionicand hydrophobic binding enhances adsorption of lipin particles.

Z is a water insoluble carrier. It need not be hydrophilic so long asthe linking groups render it water wettable or swellable aftersubstitution with the pendant hydrophobic ligands. Also, the carrierconceivably may be so strongly hydrophilic and of such a molecularweight that it is water soluble before substitution. This, however, isnot preferred since separation of the adsorbent from suspension after ithas bound lipin particles is not as efficient as with highly waterinsoluble hydrophilic carriers. It is preferred that the carrier be anorganic polymer containing a high density of hydrophilic groups.

The average molecular weight of the carrier which is desirable will varywidely as it depends upon a number of factors, including the carrier'shydrophilicity as reflected by the water adsorptive capacity of thehydrophobe-unsubstituted carrier, the extent of crosslinking within thecarrier and the degree of hydrophobe substitution which is contemplated.For example, a suitable molecular weight for starch would be greaterthan that for crosslinked dextrans, which in turn would be greater thanthat needed for satisfactory performance with cellulose or nylon.Further, the average molecular weight needed for the carriers can bereduced as the degree of substitution by the pendant hydrophobes rises.Generally, the carrier will have a molecular weight of greater thanabout 1000, ordinarily about from 2000 to 2,000,000.

A principal function of the carrier is to provide physical integrity forthe adsorbent, e.g., as a formed article, fibrous mass, woven textile ora membrane. This is an important function where the adsorbent is to beused in a filtration mode. Thus, carriers which have been crosslinked,for example by bisepoxide, glutaric dialdehyde, divinylsulfone,dibromopropanol or epichlorohydrin, are useful because of their morerigid structure.

The carriers should also be nonbiodegradable. Thus such materials asglass, silica, diatomaceous earth, agarose, polyvinyl alcohol, polyvinylpyrrolidone, cross-linked dextrans, polyacrylamide, polystyrene,styrene-divinylbenzene copolymers and nylon are preferred. Lesspreferred in applications involving long-term use, because ofsusceptibility to attack by microbial enzymes, are starches, proteinsand cellulose.

When adsorbing lipins from blood or blood fractions it is preferred toemploy as carriers nylon, polyvinyl alcohol, lower alkyl esters ofcellulose, polyvinyl pyrrolidone, polyacrylamide, polyanhydroglucose orpolyacrolein. Nylon is most preferred for this purpose. On the otherhand, for water purification cellulose, polyolefins and inorganiccarriers such as glass or diatomaceous earth are preferred because oftheir comparatively low cost.

It should be noted that the carrier need not contain or be substitutedwith any hydrophilic groups at all if the linking groups bonding thependant hydrophobe to the carrier are sufficiently numerous andhydrophilic to impart water wettability to the adsorbent. However, suchan embodiment is not preferred.

The hydrophobe is always pendant, which means that it is branched fromthe carrier as a side chain. This is critical. The linking group foundintermediate the carrier and hydrophobe is, however, optional so long asthe carrier in such cases is hydrophilic. The linking group may bedispensed with, i.e., be a bond, when the hydrophobe is bound directlyto the carrier, for example by copolymerization of ahydrophobe-substituted vinyl compound with a hydrophilic comonomer, orby radiation grafting. However, most of the convenient techniques forlinking the hydrophobe to a carrier will deposit a hydrophilic residuebetween the hydrophobe and the carrier. Such linking groups include oneor more of the groups oxo or thio ether, amido, ester, carboxyl,sulfonate, sulfone, imido, hydroxyl, thiourea, azo, silane, and amino(primary, tertiary or quaternary). Generally the hydrophilic linkinggroups will range about from 5 to 50 Å in length and have a molecularweight of about from 25 to 1000. Preferably, the group is about 10 Ålong and has a molecular weight of about 200.

Either the carrier or the linking group must be hydrophilic, but thenature of the groups which provide the hydrophilic nature may varyconsiderably. They are conveniently placed into three functionalcategories: substantially nonionogenic, weakly ionogenic and stronglyionogenic. The category used will largely depend upon the desiredfunction for the adsorbent, i.e. the solvent and lipin particlesexpected to be encountered.

Substantially nonionogenic substituents are defined for the purposesherein as those which have a pK of greater than about 12. Ordinarily,substituent groups such as hydroxyl, amido, ester, ether or silane willfall in this category. Carriers and linking groups which contain or arecomposed of these groups are preferably used to adsorb lipin particlesfrom protein-containing fractions, from solutions intended fortherapeutic administration, or from alcoholic beverages. They are alsopreferred when the physical integrity of lipin vesicles is to bemaximized, i,e., in enzyme reactors and tissue culture. For such usesthe preferred embodiments are polyamide or polyhydroxylated carriers,e.g., cellulose, nylon or polyvinyl alcohol, bound to the hydrophobethrough an ether linking group.

Where maximum adsorption of lipin particles is desired, and whereparticle rupture or disintegration and changes in the ionic compositionof the product are not so important then the carrier or linking groupmay contain or be composed of weak or strong ionogenic substituents.Weak substituents have a pK of about from 2 to 12. These are usuallycarboxyl, phosphoryl, or primary, secondary or tertiary amino groups.

Strong substituents have a pK of less than about 2. Examples aresulfonate or quaternary amino substituents. These substituents areparticularly useful in water treatments because of a lipid structurebiocidal effect that is similar to that of detergents in solution. It ispreferred to use the hydroxide form of the quaternary amine as it willnot contribute metal ions to be product. The acidic hydrophobic resinsshould be charged with pharmaceutically acceptable ions such as sodiumor potassium.

The location of the ionogenic groups is not critical. However, they areoptimally substituted immediately adjacent the pendant hydrophobicgroup, i.e., within about 10 Å. This is conveniently accomplished byproviding linking groups which are themselves ionogenic, polyfunctionalsubstituents. As examples, sulfonyl, tertiary or quaternary amino orphosphoryl groups may be linked through one functionality to the carrierand then substituted at least once with a hydrophobic group. In suchcases the hydrophobic group is preferably normal alkane of 6, 8, 10 ormore carbon atoms, up to about 20 carbon atoms, so as to minimize sterichinderance or simultaneous hydrophobic and ionic bonding of the lipinparticle. Such linking groups are preferably employed with nonionogenicor weakly ionogenic hydrophilic carriers, or with hydrophobic carriers.

Disulfide or thioester linking groups are particularly useful becausethese groups may be cleaved, respectively, by reduction withdithiothreitol or by hydrolysis at pH 11.5 for 15 minutes. Thus anyadsorbed particles can be recovered for further use or for destructionand the adsorbent then regenerated by reforming the labile linkage withfresh hydrophobe.

In a further embodiment, hydrophilic, pendant hydrophobe-substitutedpolymers may be adsorbed or covalently linked to other polymers. Thisenables the artisan to more carefully control the macroscopic characterof any formed articles made from the adsorbents of this invention, inparticular to improve their hydraulic shear resistance. It alsomultiples sites for binding the hydrophobes. For example,dextran-substituted glass may be prepared and covalently linked tohydrophobic moeities in accordance with known techniques. In such casesthe composite macromolecule functions as the carrier of this invention.

It is within the scope of this invention to employ a plurality ofdifferent hydrophobes, linking groups and carriers and ionogenic groupswithin the same matrix. Where amphoteric adsorbents are employed, i.e.,adsorbents substituted by both positively charged and negatively chargedgroups, all of which ordinarily have terminal hydrophobes, it ispreferred that the populations be segregated into mosaics in the matrixrather than being substituted adjacent to one another on the samecarrier. This is easily done by preparing differently charged matricesseparately in finely divided form, followed by mixing.

Examples of suitable adsorbents which are contemplated are set forth inTable 1 below.

                                      TABLE 1                                     __________________________________________________________________________    Representative Adsorbents                                                     d > 2                                                                         __________________________________________________________________________       ##STR2##                                                                      ##STR3##                                                                      ##STR4##                                                                      ##STR5##                                                                      ##STR6##                                                                      ##STR7##                                                                      ##STR8##                                                                      ##STR9##                                                                      ##STR10##                                                                  10.                                                                              ##STR11##                                                                     ##STR12##                                                                     ##STR13##                                                                     ##STR14##                                                                     ##STR15##                                                                     ##STR16##                                                                     ##STR17##                                                                     ##STR18##                                                                     ##STR19##                                                                     ##STR20##                                                                  20.                                                                              ##STR21##                                                                     ##STR22##                                                                     ##STR23##                                                                     ##STR24##                                                                     ##STR25##                                                                     ##STR26##                                                                     ##STR27##                                                                     ##STR28##                                                                     ##STR29##                                                                     ##STR30##                                                                  30.                                                                              ##STR31##                                                                     ##STR32##                                                                     ##STR33##                                                                     ##STR34##                                                                     ##STR35##                                                                     ##STR36##                                                                     ##STR37##                                                                     ##STR38##                                                                     ##STR39##                                                                     ##STR40##                                                                  40.                                                                              ##STR41##                                                                  __________________________________________________________________________

These adsorbents may in general be made by known processes, principallyeither by copolymerization of hydrophobe-substituted monomers withhydrophilic monomers or by linking the hydrophobes to carriers which arehydrophobic, or which become so by virtue of the linking group residueswhich contain polar groups. It is preferred to use hydrophilic carriersand to accomplish the hydrophobe linking by use of mild and well-definedlinking techniques such as the well-known carbodiimide, cyanogen halideor bisoxirane linking techniques; the reactions are mild and welldefined, and the products are stable. A myriad of other suitablesynthetic procedures will be readily apparent to the artisen.

The above described adsorbents may be in the physical form of gels;porous films having single or multiple layers; hollow microspheres;solid particles; woven matrices; compressed, randomly aligned fibrousmats; fibrous plugs; or suspensions which may in turn be precipitated byfloculating agents, collected on coarse filters or separated bycentrifugation. Fibrous mats are preferred.

The adsorbents are typically used by draining the suspension to bepurified through an adsorbent membrane, mat or column packed withparticles or fibers of the adsorbent. This is preferred over thealternative of simply admixing suspension and adsorbent in bulk and thenremoving the adsorbent by filtration or centrifugation. Compressed mats,woven matrices or membranes are most suited to situations where theadsorbent will be stressed, while loose, randomly arranged fibrousmasses are satisfactory for low pressure embodiments.

A wide range of suspensions may be treated in accordance with thisinvention. The suspending fluid need not be an aqueous solution but mayalso be a gas such as air. For example, aerosols of anhydrous lipinparticles, oil droplets or aqueous suspensions may be freed of thesuspended matter by passage through the adsorbents described herein.

The liquid suspension may also contain lipophilic proteins such asalbumin or moderate concentrations of water-miscible organic solventssuch as ethanol without significant adverse affect on adsorbentperformance. Generally less than 30% v/v of organic solvent in water isacceptable.

The particles to be separated from suspension may be oil droplets,oil-in-water emulsions, viruses, lipin vesicles such as cellularmicroorganisms, liposomes, animal cells (particularly blood cells)chylomicrons and mixtures of these particles.

The nature and concentration of the particles are not critical but willinfluence the selection or particular hydrophobe adsorbents and theamounts thereof to be used. The anticipated particle size will bear onthe average pore diameter selected for the adsorbent matrix. Generally,the average pore diameter should be about from 1.5 to 10 dwell time andquantity of adsorbent will be unique to each procedure, both parametersare readily determinable by the artisan by simply varying the quantityof adsorbent and the contact time of the suspension to arrive at anoptimal separation.

The adsorbents may be used in conjunction with separate filters whichact primarily by a sieving mechanism. For example, large, non-lipinparticles may first be removed from a crude, bacteria-containingsuspension by passage through a conventional depth filter first,followed by the adsorbent described herein, and finally a 0.2 micronpore diameter filter. Thus even though a sterile filter is used one mayemploy a considerably smaller surface area than heretofore feasiblebecause the bacterial load is reduced or eliminated by the adsorbent,thereby essentially relegating the sterile filter to an insurance role.

Hydrophobe adsorbents may be employed to separate viruses and cellularmicroorganisms from drinking water, sewage effluent, parental solutions,pharmaceuticals, alcoholic and non-alcoholic beverages. They are usefulin diagnostic assays which require the removal of cells or chylomicronsfrom test samples. They may be employed in synthetic procedures usingmicroorganisms, for example harvesting bacteria or viruses fromsuspension culture as well as aiding in the cultivation of tissuecultures. Finally, they are effective in removing lipin particles fromaerosols. times the average diameter of the particles to be removed fromsuspension. If a mixture of particles is to be filtered the largestparticle should determine the pore diameter. However, layered adsorbentshaving decreasing pore size may be employed satisfactorily.

It is not essential that the ionic strength or pH of the suspension bemodified before contact with the adsorbent, although optimal results areobtained when the adsorbent polar groups, if any, are exposed. Thus,suspensions contacted with cation substituted adsorbents should be at aslightly basic pH, and vice versa for anion substituted adsorbents.However, it may be desirable to thoroughly elute the adsorbent with aprewash of solution having similar ionic content to the suspension to bepurified, particularly if ion exchange phenomena are to be avoidedduring small scale preparative procedures for labile substances such asproteins, or where the ion exchange capacity of the adsorbent could bedeleterious to or change the ionic composition of the final product,e.g. as in the case of parenteral salt solutions.

Both the period of time for the suspension to remain in contact with theadsorbent and the comparative amounts of adsorbent and suspension willdepend upon the comparative avidity of the adsorbent for the particlesof interest in the particular suspending fluid used, the presence andrate of adherence of competitive lipin particles or lipophiles, theparticle contamination load and the pore size and hydrophilicity of theadsorbent matrix. Thus while the All cellular microorganisms are capableof adsorption to a lesser or greater degree by the hydrophobe adsorbentsdescribed herein. Examples include mycoplasma and organisms of thefamilies Pseudomonadaceae, Micrococcaceae, Lactobacillacea,Corynebacteraceae, Achromobacteraceae, Enterobacteraceae,Parvobacteraceae, Spirochaetaceae and Treponemataceae. Animal virusessuch as cytomegalovirus, herpes virus group, influenza and rubella arealso bound, as are yeasts.

The purpose of the adsorbents in sewage treatment is primarily to reducerather than eliminate the potentially infectious bioburden. Hence, flatsurfaced adsorbents or matrices having large pore sizes in the range of100-750 microns are preferable because of the large bulk of suspendedparticles typically encountered; a proportion of these particles, forexample cellulose fibers, inorganic particles and the like, arerelatively innocuous and need not be retained. The carrier and linkinggroups should not be susceptible to hydrolysis or other deleteriouschanges brought on by enzymes found in sewage; polyolefin carriers arepreferred over polysaccharides for this reason.

Hydrophobe adsorbents are effective in removing viruses and bacteriafrom drinking water. Hence it is preferred to use adsorbents which carrystrong ion exchange functions, particularly positively charged groupssuch as quaternary ammonium, and that the ion exchange function besituated adjacent to the hydrophobe. Ideally, the hydrophobe iscovalently bonded either directly into the ion exchange group or to anatom to which the ion exchange group also is bound. The carrier need notbe hydrophilic but it is desirably substituted with weakly polar groupsas described above. Hydroxy-substituted polymers are preferred. Sincethe identity of the viral and bacterial contaminants is usually unknownit is preferred to employ a mixture of discrete adsorbents, some ofwhich carry exposed positive charges and others of which have exposednegative charges. Also suitable are amphoteric adsorbents, where theboth positive and negative charges are present in the same hydrophilicmacromolecule. Where the adsorbent is employed in divided form ratherthan a formed article it is generally unnecessary to aid the separationof suspended adsorbent from suspension by use of floculating agents.

Hydrophobe adsorbents may also be used to entrap microorganisms intherapeutic solutions to be administered to animals, whether duringmanufacturing of the solutions or in their administration. Thesesolutions are predominantly parenteral and peritoneal dialysis solutionscontaining salts, carbohydrates or proteins, for example saline, aminoacids solutions, KCl solutions, 5% dextrose, and blood protein fractionssuch as antihemophilic factor, prothrombin complex, albumin, activatedprothrombin complex, insulin, hemoglobin and plasma protein fraction.Antihemophilic factor (AHF) concentrates typically contain AHF atgreater than 3 times the activity in normal plasma per unit weight ofprotein. Hydrophobe adsorbents are especially useful where thetherapeutic solution is labile to conventional sterilizing agents suchas heat and ethylene oxide, or contains a hydrocolloid. Examples of theformer include some antibiotics, amino acid and carbohydrate mixtures,proteins and polypeptides, while the latter include proteins, dextrinsand cellulose ethers.

The selection of hydrophobe adsorbent and its physical conformation willprimarily depend upon the therapeutic solution to be filtered. It isgenerally preferred to use adsorbents having substantially nonionogeniccharacter--particularly with protein-containing parenterals. However,strongly ionic adsorbents containing physiologically acceptable ions aresuitable for use with most therapeutic solutions where the adsorbent hasbeen prewashed with a representative sterile aliquot of the solution,thereby neutralizing the ion exchange activity with respect to thatparticular solution. However, there may be certain instances where theion exchange will be beneficial, for example demineralization ofcarbohydrate solutions using amphoteric exchangers. Here ion exchangeand hydrophobe adsorption activity can be advantageously combined intoone processing step. Finally the exposed polar groups of the adsorbentshould carry the same charge as the net charge of the therapeuticsolute.

The intended mode of administration of the therapeuticsolution--peritoneal, intravenous infusion, injection or oral--generallyis not material to the selection of adsorbent at the manufacturinglevel. Table 1 adsorbents 1, 3, 8 and 16 are preferred, with theadsorbents 1 and 3 most preferred. They are most advantageously employedas membranes, woven fabrics or random, fibrous masses having an averagepore diameter of about from 0.75 to 20 microns, preferably about from1.5 to 10 microns.

The same adsorbents may be used when administering the solutions topatients. This is conveniently accomplished by including the adsorbentsin administration sets. These sets usually include (a) a conduitterminating at one end with a means for connecting the conduit to acontainer of the solution and at the other end with a means for enteringthe body of the animal and (b) a filter interposed in said conduitbetween said both means.

The means for entering the body include needles, and venous orperitoneal catheters. Flow control devices and connectors for themultiple attachment of parenteral solution containers are frequentlyincluded in such sets. Hydrophobe adsorbents can be used in place of thefilter or interposed between the filter and the solution container as anadjunct to the filter. The hydrophobe adsorbent is preferably the solefilter when protein-containing parenteral solutions such asantihemophilic factor are to be administered, because the average poresize may be increased to about from 2 to 20 microns from the usual 0.2to 0.5 microns, thereby increasing the filter flux. Further, air blockformation is reduced by the hydrophobe adsorbents when compared to thewholly hydrophilic filters previously employed. Thus the complex dualfilters which have been proposed to ameliorate this problem may bereplaced by unitary, hydrophobe absorbent filters.

The capacity of the hydrophobe adsorbents for hepatitis is generallysuperfluous when treating blood protein-containing pharmaceuticals. Thestarting plasma has been screened for assayable hepatitis and, in thecase of products such as albumin, heat treated to destroy the virus.Other parenteral solutions are free of hepatitis virus because nopotentially infective substance is used as a starting material. However,while such products are essentially free of assayable hepatitis it isdesirable to remove other viruses that may be present and are notscreened for, e.g. herpes and rhinoviruses.

The hydrophobe adsorbents are also useful in the pasteurization ofalcoholic beverages, primarily beer and wine. Such products aredifficult to pasteurize in a manner which does not also deleteriouslyaffect the beverage quality. Surprisingly, it has been found thatethanol concentrations in aqueous solutions of up to about from 0.5% to30% do not significantly interfere with the capacity of the hydrophobeadsorbents to bind yeast and bacteria suspended in such solutions.

In accordance with this invention, beer and wine ordinarily arepasteurized by simply passing the fermentate through a matrix ofhydrophobe adsorbent. The average pore diameter here will be larger thanwith filters having the primary task of removing bacteria because theyeast cells are comparatively larger. A suitable pore diameter rangesabout from 3 to 20 microns. Ion exchange adsorbents may be used, keepingin mind the caveats expressed above regarding parenteral solutions. Thesame embodiments as were discussed above in connection with hydrocolloidsolutions are satisfactory in pasteurizing alcoholic beverages.

The preceding discussion has focused on microbes as contaminants, wheretheir removal ordinarily is followed by their destruction. Thehydrophobe adsorbents, however, also are extremely useful in recoveringcells from suspension culture or mammalian cell culture. The adsorbentsare most useful in the first embodiment. Here a microbe, generally abacterium, is cultured in suspension in a nutrient solution, ordinarilythrough the end of the log growth phase. Then a substrate solution isapplied to a column of hydrophobe adsorbed organisms and product drawnoff as column eluate. Preferably the solution contains no general growthfactors such as carbohydrates or nitrogen sources. Products which may bemanufactured by this technique or by fluidized bed fermentations includefructose, various amino acids, nucleotides, penicillin, andstaphylococcal protein A.

Surface adhering cells such as mammalian cell lines from disaggregatedorgans also may be cultured simply by agitating finely divided adsorbentin a nutrient medium inoculated with the cells. The cells are separatedfrom suspension by centrifuging or filtering the adsorbed cells. Theymay then be used in the same way as bacteria supra or in applicationsunique to animal cells, e.g., artificial organs or in the synthesis ofunique products such as antibodies by hybridoma cells and viruses forvaccine production.

The adsorbed lipin particles, whether cells, liposomes or viruses, alsocan be desorbed for their recovery or for regeneration of the adsorbent.This may be accomplished by (a) cleaving the linking group as describedabove, (b) introducing solvents having nonpolar groups in place of or asa substantial proportion of the eluting solvent, or (c) adding otherlipin particles to displace the adsorbed materials. For example, virionsmay be recovered for vaccine preparation or other uses by eluting theadsorbent with an aqueous solution of salt and a high concentration,i.e., a greater than 30% v/v, of a lipophilic solvent such as ethyleneglycol, acetone, ether or alkanol. Recovery of the adsorbent isfacilitated if the solvent is also water soluble. Forty percent ethyleneglycol in saline is preferred. Alternatively, a saline suspension ofliposomes prepared in known fashion may be passed through the matrix andthe virions recovered from the aqueous phase after removing the elutedliposomes, e.g. by centrifugation or extraction of the liposomes into animmiscible solvent.

Finally, hydrophobe adsorbents greatly facilitate diagnostic assays foranalytes in lipemic samples of plasma or serum. The opaque, milkyappearance of such samples and the difficulty with their use is opticalassays is largely a function of chylomicrons. The chylomicrons can beadsorbed simply by passing the sample through a matrix of hydrophobeadsorbent, preferably in conjunction with a serium skimmer. Serumskimmers are disclosed in U.S. Pat. Nos. 3,799,342 or 3,865,731. Theirsalient features are a filter for removing the insoluble constituents ofserum and a chamber for collecting filtered serum or plasma. They areused to skim serum from collection tubes in which collected bloodsamples have been allowed to clot. These devices generally comprise acollection receptacle for filtered sample, a valve which permits thesample to flow into the collection receptacle but not in the oppositedirection, a filter disposed on the opposite side of the valve from thereceptacle and a flexible sealing member for engaging the inner surfacesof the test sample container. The device is used by pushing it into thetest sample container, usually a collection tube containing clottedblood. The sealing member prevents passage of fluid between the skimmerand the walls of the collection tube. Instead, the sample flows throughthe filter and one-way valve into the receptacle. The improvement ofthis invention comprises using a hydrophobe adsorbent in such devices asthe filter, or as an element thereof. Preferably the entire filter isnonwoven plug or mat of fibrous hydrophobe adsorbent having an averagepore diameter from about 15 to 100 microns. A large pore diameter ofabout 75 microns is considered optimal because the rapid passage ofplasma or serum facilitated by such an open network reduces theadsorption of high density and low density lipoproteins. Smaller porediameters are acceptable if lipoprotein assays are not contemplated. Thesample may then be assayed for an analyte using a procedure in whichchylomicrons ordinarily would interfere, for example in optical assaysas defined above.

The hydrophobe adsorbents are particularly useful for screeningaerosol-borne lipin particles from air. The maintenance of sanitary, orparticle-free atmospheres in hospital operating rooms, sterile productmanufacturing and electronics assembly operations is paramount.Hydrophobe adsorbents are effective in screening oil droplets andmicroorganisms from the air supplies used in such environments, whetheror not the aerosols contain water. The adsorbents may be employed withconventional passive filters to collect any nonlipin materials. Theadsorbents may be regenerated by washing thoroughly with a volatileorganic solvent or by hydrolysis or reduction of linking groups asdescribed above.

The invention will be more fully understood upon reference to thefollowing Examples.

EXAMPLE 1

Preparation of adsorbent 1, Table 1. 5 g of nylon wool (sold by theFenwal Division of Travenol Laboratories for the affinity collection ofgranulocytes) was immersed in a 400 ml of an emulsion containing 350 mldistilled water and 50 ml of 1,4-butanediol diglycidyl ether and stirredfor 2.45 hours. After decanting, but without rinsing, 100 ml octylaminewere added and stirred for one hour. Both reactions were conducted at22° C. A white, oily precipitate was eluted from the substituted nylonunder extensive washing with distilled water. The product was soft andfelt oily to the touch. A 20 ml syringe was packed to the 5 ml mark withadsorbent and rinsed exhaustively. The effect of an ethanol rinse wasevaluated by rinsing a packed syringe with 30 ml ethanol followed by 30ml of distilled water. The ability of the adsorbent to removed bacteriawas assayed by adding 25-30 ml of contaminated normal serum albumin tothe syringe and allowing the albumin to passively flow through thefilter. This albumin was heavily infected with flora indigenous to theblood plasma fractionation facility from which it was obtained,primarily thought to be Pseudomonas sp. in populations greater than 10⁶organisms/ml. The last 5 ml were collected, diluted as indicated andaliquot plated onto culture media. The results are shown in Table 2below.

                  TABLE 2                                                         ______________________________________                                                Adsorbent    Eluted Bacteria/ml                                       ______________________________________                                        water washed                                                                            none           TNTC* at 1:100 dilution                                        unsubstituted nylon                                                                          TNTC at 1:100 dilution                                         modified nylon  0                                                   ethanol washed                                                                          unsubstituted nylon                                                                          TNTC at 1:100 dilution                                         modified nylon 32                                                   ______________________________________                                         *Too numerous to count.                                                  

EXAMPLE 2

Preparation of adsorbent 3, Table 1. 5 g of nylon wool from the samesource as Example 1 was immersed into 400 ml of an emulsion containing350 ml distilled water, 5.5 g N,N¹ -dicyclohexylcarbodiimide and 50 mlof octanoic acid, then reacted for 1 hour at ambient temperature whilestirred. The firm, modified nylon was washed with distilled water, butunlike Example 1 no precipitate eluted. An ethanol washed column wasalso prepared as in Example 1, and the bacterial retention qualitiesevaluated in the same fashion with contaminated normal serum albumin.Bacterial adsorption was satisfactory.

EXAMPLE 3

The performance of the Example 1 modified nylon was evaluated usingadditional suspending fluids and fully characterized contaminantorganisms. The organisms designated in Table 3 below were seeded at theindicated populations into sterile phosphate buffered saline (PBS), 5%normal serum albumin (NSA), distilled water, pasteurized whole milk andbeer (Coors). Plugs of modified and unmodified (control) nylon wereplaced into a 12 ml syringe and washed with 15 ml of sterile heartinfusion broth. Three ml of each contaminated liquid were then passedthrough the syringes and the eluate bacterial population determined bypromptly plating the eluate onto culture media in conventional fashion.The results are reported in Table 3.

                  TABLE 3                                                         ______________________________________                                                   Bacteria/ml                                                        Medium Organism  Inoculum Control Eluate                                                                           Test Eluate                              ______________________________________                                        PBS    S. aureus 320,000  72,000    1                                                P. diminuta                                                                             120,000  24,000    0                                         Water  S. aureus 330,000  39,000    3                                                P. diminuta                                                                              50,000  41,000    0                                         Milk   S. aureus 480,000  67,000    24,000                                           P. diminuta                                                                             140,000  33,000    32,000                                    Beer   S. aureus 440,000   8,000    140                                              P. diminuta                                                                              80,000  29,000    0                                         NSA    S. aureus 280,000  69,000    2                                                P. diminuta                                                                              80,000  39,000    0                                                B. subtilis                                                                              30,000  11,000    90                                        ______________________________________                                    

It was concluded from Table 3 that hydrophobe adsorbents are capable ofmultifold reduction or elimination of the microbial burden of a widevariety of contaminated liquids. This effect is accentuated byelectrostatic or mechanical seiving by the nylon matrix as evidenced bythe lowered populations in the control eluates. The small reduction inmilk contamination is believed to be the result of competitive bindingof the hydrophobes by lipid vesicles and residual flora endogenous tothe milk after pasteurization; an increased amount of adsorbent wouldremove both of these components as well as added bacteria. Parallelexperiments with a yeast, Saccharomyces cerevisiae also were conducted.The modified nylon was somewhat more effective than the control inremoving the contaminants from PBS, NSA, water and beer, but with milkthe control was effective as the modified nylon. This was attributed tothe likelihood that the yeast was considerably larger than bacteria andpredominately filamentous, therefore mechanically retained by the nyloncontrol.

EXAMPLE 4

Satisfactory removal of E. coli from saline suspension could be achievedupon repeating Example 3 with granules of adsorbent 34 described inTable 1 above.

EXAMPLE 5

Satisfactory removal of Streptococcus faecalis (predominantly diploid)in serum broth could be achieved upon repeating Example 1 with 10-50U.S. mesh beads of adsorbents 20,21,25,29 or 30 described in Table 1above.

EXAMPLE 6

Mycoplasma prepared in known fashion and suspended in isotonic nutrientbroth could be satisfactory adsorbed upon passage through adsorbent 16,Table 1, in the form of five layers of cotton cloth.

EXAMPLE 7

Adsorbent 4 prepared from hardwood sawdust and sandwiched at a thicknessof 200 mm between two fine-mesh stainless steel screens could removeendogenous microorganisms, primarily coliforms, from raw sewage.

EXAMPLE 8

Acanthamoeba castellani, a soil amoeba, could be removed from a growthmedium containing 1.5% glucose and 1.5% proteose peptone by passage ofthe suspension through a cotton fiber plug of adsorbent 9, Table 1.

EXAMPLE 9

An aerosol of a saline suspension of E. coli produced by a householdvaporizer could be screened from a 1:100 dilution in humidified air byblowing the aerosol at a rate of 1 cubic foot/min through a 5×15 cmcolumn packed loosely with a adsorbent 3, Table 1, in the form ofmodified nylon fibers.

EXAMPLE 10

5 ml of lipemic human serum could be clarified by the conventional useof an Accu-sep serum skimmer in which the filter was replaced byadsorbent 4, Table 1, in the form of a matted cellulose fiber plug.

EXAMPLE 11

Phospholipid vesicles were prepared according to the procedure of Batzriet al., "Biochem. Biophys. Acta" 298:1015-1019 (1973) using diacetylphosphate to render the bilayer vesicles negatively charged. Thevesicles could be adsorbed by briefly mixing adsorbent 11 of Table 1with the suspension and centrifuging.

EXAMPLE 12

This example is concerned with the continuous manufacture ofstreptokinase from immobilized streptococci. A 5 liter column was packedwith polyacrylamide beads modified as adsorbent 21, Table 1. Hemolyticstreptococci were cultured as described in U.S. Pat. No. 3,855,065 and a1 liter inoculumn of log phase organisms introduced onto the column,followed by a steady flow of 75 ml/hour of a nutrient medium containing8% corn steep liquor and 7% Cerelose in water. The column eluate wascollected for 24 hours and the streptokinase recovered by precipitationwith ammonium sulfate. The columns would function satisfactorily untilmicrobial replication clogged the pores and restricted flow or until thecolumn adsorptive capacity became overloaded and effluent break outoccurred. The column lifetime may be extended by substituting saline fornutrient medium after 3 hours' cultivation.

EXAMPLE 13

Example 2 of U.S. Pat. No. 3,821,086 is followed by the manufacture offructose from glucose except that the Primafloc C-7 employed by thepatentee to insolubilize the Arthrobacter species was substituted byadsorbent 17, Table 1, as modified course hardwood sawdust. When enzymeactivity declined to an unacceptable level the adsorbent could berecycled by reducing the adsorbent with an aqueous solution of 0.5 Mdithiothreitol and 0.001 M EDTA for 2 hours, washing the adsorbentextensively with distilled water, and reacting the adsorbent withdioctyldisulfide under oxidizing conditions regenerate the adsorbent.

EXAMPLE 14

Adsorbent 3, Table 1, as a modified nylon wool was loosely packed into apolyethylene flattened cylinder containing 3 cc volume. This filterassembly could be satisfactorily used in the continuous ambulatoryperitoneal dialysis equipment disclosed in copending U.S. patentapplication Ser. No. 27,419 as an adjunct to or in place of theparticulate filter 26 described in that application.

EXAMPLE 15

Herpes virus type 1, strain hf was recovered from MA104 cell culture andthe suspension adsorbed upon passage of 5 ml of suspension through a 1ml volume plug of either of the adsorbents employed in Example 1 or 2.Adsorption was evaluated by serially diluting the adsorbent eluate,inoculating monolayer MA104 cell cultures with the dilutions andobserving for cytopathic effects.

EXAMPLE 16

1.5 g of nylon fibers were packed into a 1-inch diameter filter holder,mixed overnight with about 15 ml of concentrated acetone solution ofbis-epoxide. Then an aqueous solution of linear dextran (average 200,000D) at saturation is mixed with the activated fibers for 24 hours at pH9. The product is rinsed with copious quantities of cold distilled waterand brought to dryness with 50%, 70%, and then 95% acetylnitrilefollowed by acetone. Then 100 mg cyanogen bromide in ice cold water (pH9) is mixed slowly with the modified nylon and the pH maintained withNaOH. When no more NaOH was required, a saturated solution of n-octylamine is mixed overnight with the activated matrix. The following day,the system is washed extensively with water and then 0.5 M ethanolamine.A suspension of 1×10⁵ Pseudomonas diminuta organisms in demineralizedwater was allowed to percolate through the filter. The bacteria weresatisfactorily adsorbed.

I claim the following:
 1. A method for removing lipin particles fromsuspension in a fluid, comprising contacting the suspension with anadsorbent composition having the formula

    [(Y).sub.e B].sub.d Z

wherein Y is a hydrophobic ligand, B is a strong ionogenic group, Z is awater insoluble carrier, e is an integer and d is greater than 2 underconditions to adsorb the particles, followed by separating thecompositions from the fluid.
 2. The composition of claim 1 wherein Y isa group having the formula ##STR42## and wherein R is hydrogen, nitro,alkyl, alkyl ether, halogen, monocyclic aromatic hydrocarbon or acarbocycle system;A is a bond, monocyclic aromatic hydrocarbon orcarbocycle system; b is an integer; J is oxygen, sulfur or a bond; and nand y are zero or an integer;with the proviso that where A is a bond andR is hydrogen, nitro or halogen then the sum of n and y is an integergreater than
 5. 3. The composition of claim 2 wherein R is alkyl havingabout from 1 to 6 carbon atoms.
 4. The composition of claim 2 whereinthe sum of n and y is 8, R is hydrogen, J and A are bonds and e and bare both
 1. 5. The composition of claim 1 wherein Y is normal alkylhaving from 6 to about 20 carbon atoms.
 6. The conposition of claim 1wherein Y is phenyl.
 7. The composition of claim 1 wherein B and Ztogether are hydrophilic.
 8. The composition of claim 1 wherein Z is anorganic polymer.
 9. The composition of claim 8 wherein the polymercontains strong ionogenic groups which are not substituted with ahydrophobic ligand.
 10. The composition of claim 9 wherein the group issulfonyl or quaternary amino.
 11. The composition of claim 1 wherein Zis inorganic.
 12. The composition of claim 11 wherein Z is glass. 13.The composition of claim 1 wherein e is
 1. 14. The method of claim 1wherein the fluid is an aqueous ligand.
 15. The method of claim 14wherein the liquid has an ionic strength of less than about 0.1.
 16. Themethod of claim 15 wherein the liquid contains about from 1 to 30%protein (w/v).
 17. The method of claim 14 wherein the particles areviruses.
 18. The method of claim 1 wherein the adsorbent is a formedarticle.
 19. The method of claim 1 wherein the adsorbent is woven or amembrane.
 20. The method of claim 1 wherein the adsorbent is a fibrousmass.
 21. The method of claim 1 wherein Z is an organic polymer and dranges about from 0.5 to 0.1 times the number of monomer unitsconstituting the polymer.